Sequence analysis of saccharide material

ABSTRACT

A method of analyzing and sequencing saccharide material composed of saccharide chains. The saccharide chains are end referenced, e.g., by labeling or tagging at their reducing ends, and the saccharide material is subjected to a controlled partial depolymerisation using a selection scission reagent, for example, low pH nitrous acid, which cleaves internal glycosidic linkages in accordance with known linkage specificity so as to produce a mixed set of saccharide chain fragments having different lengths ranging throughout the full spectrum of possible lengths for the particular glycosidic linkage specificity of the selective scission reagent employed. Samples of the mixed set of saccharide chain fragments are then treated with selected exoenzymes including exoglycosidases that cleave only particular glycosidic linkages at the non-reducing end of saccharide chains. These exoenzymes are applied to the samples either singly or in combination in accordance with a predetermined strategy. The treated samples are then analyzed, to detect the chain fragments present which have a reducing end derived from the reducing end of the corresponding chain in the original saccharide material and the monosaccharide sequence in the saccharide starting material is then deduced.

FIELD OF THE INVENTION

The present invention is concerned with sequence analysis of saccharidematerial and it is especially applicable to the sequencing of saccharidechains containing numerous amino sugar residues such as, for example,are found in glycosaminoglycans (GAG's) which include the biologicallyimportant polysaccharides, heparan sulphate (HS) and heparin.

BACKGROUND

Heparan sulphate (HS) and heparin are chemically-related linearglycosaminoglycans (GAG's) composed of alternate α,β-linked glucosamineand hexuronate residues with considerable structural variation arisingfrom substitution with acetyl and N- and O-sulphate groups, and from thepresence of D- and L-isomers of the hexuronate moieties. Thesepolysaccharides are of fundamental importance for many diverse cellularand biochemical activities. Their regulatory properties are dependent ontheir ability to bind, and in some cases to activate, protein moleculeswhich control cell growth, cell adhesion, and enzyme-mediated processessuch as haemostasis and lipid metabolism. However, analysis ofprotein-binding monosaccharide sequences in HS/heparin is generallydifficult and a universal procedure suitable for routine use has notbeen described to date.

An object of the present invention is to provide a new method ofsequence analysis of saccharide fragments such as oligosaccharides thatmay be derived from HS (or heparan sulphate proteoglycan HSPG) andheparin, this method enabling rapid elucidation of recognition sites andother sequences of interest and thereby facilitating the rational designof synthetic compounds to serve as drugs for therapeutic modulation ofpolysaccharide function.

SUMMARY OF THE INVENTION

In one aspect the invention may be regarded as being based on a conceptof bringing about a preliminary partial depolymerisation by scission ofspecific intrachain linkages in reducing end referenced saccharidechains, such as for example HS/heparin saccharide chains, followed byexoenzyme removal of non-reducing end (NRE) sugars or their sulphategroups so as to yield a range of labelled fragments that can beseparated by gel electrophoresis or other appropriate techniques to givea read-out of the sequence of sugar units and their substituents.Although the invention may be described mainly in relation tosaccharides that are found in heparan sulphate and heparin, the basicprinciple of the sequencing strategy is applicable to many other GAG'sand different saccharides, including the saccharide component ofglycoproteins.

Use of exoenzymes, in particular exoglycosidsases, for removal ofterminal sugar residues at the non-reducing end of saccharide chains haspreviously been proposed in connection with methods for sequencing suchchains, for instance in WO 92/02816 and in WO 92/19974 and WO 92/19768.However, in these prior art proposals there has been no preliminary stepof partial depolymerisation of the saccharide material, involvingcleavage of internal glycosidic linkages, before treatment with saidexoenzymes. In WO 92/02816 for example, it is proposed in relation to asaccharide sequencing method disclosed therein to use exoenzymessuccessively to remove and identify terminal sugar residues at thenon-reducing end of initially undegraded saccharide chains, and to carryout a series of sequential steps with the residual saccharide materialbeing recovered after each step before proceeding to the next. In WO92/19974 and WO 92/19768, although exoenzymes are mentioned inter aliaas possible sequencing agents, again it is proposed that these beapplied sequentially direct to an oligosaccharide being analysed in aniterative process without a preliminary partial depolymerisation step asrequired by the present invention. In all these prior art methods thesequencing information is obtained and presented in a different mannerto that in the present invention.

An acknowledgement is also made of a paper by Kyung-Bok Lee et al,Carbohydrate Research, 214 (1991), 155-168, which refers to the use ofexoglycosidases and of endoglycosidases in connection with sequencing ofoligosaccharides. This publication does not, however, disclose thecombined use of both exoglycosidases and endoglycosidases in sequence inthe same manner as herein defined in the claims relating to the presentinvention.

More specifically, the present invention broadly provides a method ofanalysing and sequencing saccharide material comprising saccharidechains which contain more than three monosaccharide units interconnectedthrough glycosidic linkages that are not all identical and which eachinclude a referencing feature at their reducing end, wherein selectedexoenzymes comprising exoglycosidases of known specificity that cleaveonly particular glycosidic linkages at the non-reducing end ofsaccharide chains are used to obtain sequence information, said methodbeing characterised in that it comprises the sequential steps of:

(a) subjecting said saccharide material to partial depolymerisation bycontrolled treatment with a selective scission reagent that acts inaccordance with a known predetermined linkage specificity as anendoglycosidase to cleave a proportion of susceptible internalglycosidic linkages, that is, susceptible glycosidic linkages spacedfrom the non-reducing end of the saccharide chains, thereby to produce amixed set of saccharide chains, intact chains and fragments of intactchains, having different lengths representative of the full spectrum ofall possible lengths given the particular glycosidic linkage specificityof the selective scission reagent employed,

(b) treating a selected sample or samples of said mixed set ofsaccharide chains and chain fragments with said exoenzymes, eithersingly or in combination in accordance with a predetermined strategy, toan extent sufficient to obtain complete digestion and cleave susceptiblelinkages at the non-reducing end of all the saccharide chains, and then

(c) analysing said sample or samples to detect the various saccharidechain fragments generated by the cleavage treatments which are presenttherein and which have a reducing end derived from the reducing end ofthe corresponding chain in the original saccharide material, andobtaining, collectively from the results of said analysis, informationenabling the monosaccharide sequence in the original saccharide materialto be at least partially deduced.

In carrying out this saccharide sequencing method of the presentinvention, the saccharide material will generally be treated, usuallybefore the controlled partial depolymerisation step, to modify thesaccharide chains at their reducing ends in order to introduce thereducing end referencing feature for providing a common reference pointor reading frame to which the monosaccharide sequence can be related andfor facilitating, during analysis, detection of chain fragments having areducing end derived from the reducing end of the corresponding chainsin the original saccharide material. This end referencing feature isconveniently provided by selectively labelling or tagging themonosaccharide units at the reducing ends of the saccharide chains,using for example radiochemical, fluorescent, biotin or othercalorimetrically detectable labelling means.

If low pH nitrous acid is used for carrying out the partialdepolymerisation of the saccharide material as hereinafter described, apresently preferred fluorescent labelling agent is anthranilic acid asreferred to in more detail later. However, if a selective scissionreagent other than nitrous acid is used for bringing about the partialdepolymerisation, e.g. an endoglycosidase enzyme, an aminocoumarinhydrazide, e.g. 7-amino4-methylcoumarin-3-acetyl hydrazide, may bepreferred for providing a fluorescent labelling agent having arelatively high labelling efficiency. For use as a radiochemicallabelling agent tritiated borohydride may be used.

In an alternative but usually less preferred technique for providingend-referenced saccharide chains or chain fragments, the chains may beimmobilized by coupling the reducing ends to a solid phase support. Thiscan then permit those chain fragments, produced by the partialdepolymerisation treatment, which are not contiguous with the reducingends of the original undegraded chains to be physically separated andremoved, whereupon subsequent release of the immobilized chain fragmentsfrom the solid phase support then provides the required mixed set ofchain fragments ready for exoenzyme treatment as before.

In preferred embodiments, as hereinafter more fully described,electrophoretic separation means such as polyacryiamide gelelectrophoresis (PAGE), e.g. gradient PAGE, will usually be used fordetecting the fragments produced by the cleavage treatments, thesefragments being separated according to differences in length andcomposition which are reflected in different mobilities in theelectrophoretic medium. If necessary, for uncharged or lightly chargedsaccharide chains, the material can be treated in a preliminaryoperation so as to incorporate therein suitable electrically chargedgroups in a known manner in order to permit the use of electrophoreticseparation techniques. This will not usually be necessary, however, insequencing HS or heparin oligosaccharides which already contain asignificant number of charged sulphate and carboxyl groups. Otheralternative separation techniques, for example capillary electrophoresisor high performance liquid chromatography (HPLC), may also be used fordetecting the fragments so long as the requisite resolving power isavailable.

After the controlled partial depolymerisation step the mixed set ofsaccharide chain fragments produced will usually be used to provide anumber of separate samples. One of these samples, and generally acontrol sample of the original material, will then be subjected to theseparation technique, e.g. gradient PAGE, to separate and detect thedifferent fragments present for reference purposes before exoenzymetreatment. At the same time, other samples of the set of fragments willalso be subjected to the same separation technique so as to separate anddetect the different saccharide fragments present after each of theseother samples has been treated with a different exoenzyme or combinationof exoenzymes.

In applying the invention to the sequencing of saccharide chainscontaining many amino sugar residues, such as are found inglycosaminoglycans (GAG's) for which the method is especially useful,the preliminary controlled partial depolymerisation involving cleavageof specific internal glycosidic linkages is most conveniently carriedout as hereinafter more fully described using nitrous acid at low pH asa chemical selective scission reagent. It is also possible, however, insome cases as an alternative to a chemical selective scission agent touse appropriate enzymatic endoglycosidases, e.g. the bacterial lyasesheparinase (EC 4.2.2.7) or heparitinase (EC 4.2.2.8), under suitableconditions to bring about selective enzymatic cleavage of internalglycosidic linkages.

As GAG's and similar saccharides also generally contain varioussulphated monosaccharide units, the selected exoenzymes used fortreating tile fragments obtained after the initial hydrolysis andpartial depolymerisation will usually include, in addition toexoglycosidases, selected exosulphatases for effecting a controlledremoval of particular sulphated groups from specific terminalmonosaccharide residues at the non-reducing end of the chains. Otheradditional specific enzymes may also be used in analysing the fragmentsobtained after the partial depolymerisation as part of the overallstrategy selected for extracting or confirming the sequence informationrequired.

Examples of selective scission reagents which may be used in carryingout the sequencing method of the present invention include thefollowing:

Reagents Linkage Specificity (1) *Nitrous Acid GlcNSO₃ → HexA (2)*Glucuronidase (Gase) GlcA → GlcN.R (β-D-glucuronidase) (3) *Iduronidase(Idase) IdoA → GlcN.R (α-L-iduronidase) (4) *N-acetylglucosaminidaseGlcNAc → HexA (5) ▪Iduronate-2-Sulphatase (I2Sase)

(6) ▪Glucosamine-6-Sulphatase (G6Sase)

e.g. ▪N-acetylglucosamine-6-sulphatase

(7) *Glucuronate-2-Sulphatase

(8) ▪Sulphamate sulphohydrolase GlcNSO₃ → HexA Abbreviations and labelsused above have the following meanings: GlcN. = Glucosamine R = Acetyl(Ac) or SO₃ _(⁻) HexA = Hexuronic acid GlcA = Glucuronate IdoA =Iduronate *Cleaves glycosidic linkages ▪Removes sulphate groups only

The enzymes mentioned above are exoenzymes which act specifically toremove the terminal sugar residues or their sulphate substituents at thenon-reducing end (NRE) of glycan fragments. Details of many such enzymesare readily available in the literature, and by way of example referencemay be had to an informative review article entitled “Enzymes thatdegrade heparin and heparan sulphate” by John J. Hopwood in “Heparin:Chemical and Biological Properties, Clinical Applications”, pages 191 to227, edited by D. A. Lane et al and published by Edward Arnold, London,1989, and to another review article entitled “Lysosomal Degradation ofHeparin and Heparan Sulphate” by Craig Freeman and John Hopwood in“Heparin and Related Polysaccharides”, pages 121 to 134, also edited byD. A. Lane et al and published by Plenum Press, New York, 1992.

Some of these enzymes are available commercially and others can beisolated and purified from natural sources as described in theliterature. Moreover, in some cases recombinant versions are known and,when available, these will often be preferred because of a high level ofpurity that can usually be achieved. Published papers in which theisolation and preparation or properties of some of the enzymes referredto above are described include: Alfred Linker, (1979), “Structure ofHeparan Sulphate Oligosaccharides and their Degradation by Exo-enzymes”,Biochem. J., 183, 711-720; Craig Freeman and John J Hopwood, (1992),“Human α-L-iduronidase”, Biochem. J., 282, 899-908; Wolfgant Rohrbornand Kurt Von Figura, (1978), “Human Placenta α-N-Acetylglucosaminidase:Purification. Characterisation and Demonstration of Multiple RecognitionForms”, Hoppe-Seyler's Z. Physiol. Chem., 359, 1353-1362; Craig Freemanand John J Hopwood, (1986), “Human Liver Sulphamate sulphohydrolase”,Biochem. J., 234, 83-92: Craig Freeman, et al, (1987), “Human LiverN-acetylglucosamine-6-sulphate sulphatase”, Biochem. J., 246, 347-354;Craig Freeman and John J Hopwood, (1991), “Glucuronate-2-sulphataseactivity in cultured human skin fibroblast homogenates”. Biochem. J,.279. 399-405: Craig Freeman and John J Hopwood, (1987), “Human liverN-acetylglucosamine-6-sulphate sulphatase”, Biochem. J., 246. 355-365:Irwin G. Leder (1980), “A novel 3-O sulfatase from human urine acting onmethyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulphate”, Biochemicaland Biophysical Research Communications, 94, 1183-1189; Julie Bielicki,et al, (1990), “Human liver iduronate-2-sulphatase”, Biochem. J., 271.75-86: Irwin G. Leder (1980), “A novel 3-O sulfatase from human urineacting on methyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulphate”,Biochemical and Biophysical Research Communications, 94, 1183-1189; andJulie Bielicki, et al, (1980), “Evidence for a 3-O-sulfatedD-glucosamine residue in the antithrombin-binding sequence of heparin”,Biochemistry, 77, 6551-6555.

A recombinant version of an exoenzyme and the preparation thereof isdescribed for example in connection with a synthetic α-L-iduronidase ininternational patent publication WO 93/10244.

The contents of the above-mentioned publications are incorporated hereinby reference.

The nitrous acid (HNO₂) reagent used at low pH cleaves hexosaminidiclinkages when the amino sugar is N-sulphated (GlcNSO₃) irrespective ofthe position of the linkage in the saccharide chain, but mostimportantly GlcNAc→GlcA linkages are resistant to HNO² scission. Thecontrolled hydrolysis and partial depolymerisation with nitrous acid canbe achieved by preparing the reagent as described by Steven Radoff andIsidore Danisliefsky, J. Biol. Chem. (1984), 259, pages 166-172 apublication of which the content is also incorporated herein byreference. A typical example with practical details, however, isdescribed below.

Example of Conditions for Nitrous Acid Hydrolysis and PartialDepolymerisation of Saccharides

The saccharide to be treated (1-2 nmoles) is dried down by centrifugalevaporation, dissolved in 80 μL of distilled H²O and cooled on ice. Tothis solution is added 10 μL of 190 mM HCl and 10 μL of 10 mM NaNO₂,both precooled on ice. These reactants are mixed by vortexing andincubated on ice. At predetermined time points (for example 0, 20, 40,60, 90 and 120 minutes), aliquots of the reaction mixture are removedand the low pH HNO₂ hydrolysis is stopped either by addition of excessammonium sulphamate to quench the reagent, or by raising the pH above4.0 (for example by addition of Na₂CO₃ solution). It has in fact beenfound most convenient to stop the reaction by addition of 1/4 volume of200 mM sodium acetate buffer, pH 5.0. This raises the pH toapproximately pH 4.3-4.4 and provides buffer conditions immediatelycompatible with subsequent enzyme treatments, thus avoiding the need forany further clean-up steps such as removal of salts or buffer exchanges.Finally, once all the time points are complete the aliquots are remixedand pooled. This is crucial since it creates a mixed set of saccharideproducts, hydrolysed partially and at random, which contain fragmentscorresponding to all possible cleavage positions, whereas a single timepoint would not create such a representative set. Thus, the fragmentshave different lengths ranging throughout the full spectrum of possiblelengths for the particular glycosidic linkage specificity of the HNO₂reagent, and ideally there should be a fairly even distribution of thedifferent length fragments.

In carrying out the invention, it will be appreciated that in effect thecontrolled, incomplete hydrolysis of N-sulphated disaccharides by theHNO₂ treatment, i.e. the partial HNO₂ scission or depolymerisation(herein denoted as pHNO₂), is used to “open-up” the glycan structure ofthe saccharide material under analysis so as to expose a range of NREsugars and sulphate groups to attack by specific exoglycosidases andexosulphatases. Indeed, this dual approach of combining a preliminarycontrolled hydrolysis and partial depolymerisation involving cleavage ofinternal linkages with a progressive action of exoenzymes acting at thenon-reducing end of the fragments produced can be regarded as being animportant and significant key feature characterising the sequencingmethod of this invention.

MORE DETAILED DESCRIPTION

The invention and the manner in which it may be carried out will now behereinafter described in more detail with reference to non-limitingillustrative examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In connection with the above-mentioned illustrative examples, referenceshould be made to the accompanying drawings in which:

FIG. 1A represents a hypothetical but possible structure of anoctasaccharide (degree of polymerisation dp=8) fragment that may bederived from heparan sulphate (HS) or heparin;

FIG. 1B shows the octasaccharide of FIG. 1A after partialdepolymerisation which provides a mixed set of saccharide chainfragments:

FIG. 2 is a chart or diagram illustrating the electrophoretic separationand analysis using PAGE of the set of saccharide chain fragments shownin FIG. 1B following exoenzyme treatment in accordance with theinvention: and

FIG. 3 is a photocopy of a photographic representation of aelectrophoretic gel banding pattern derived from some preliminarystudies undertaken during development of the present invention.

EXAMPLE 1

For sequencing the octasaccharide fragment illustrated in FIG. 1A of theaccompanying drawings, initially the GlcNSO³— unit at the reducing end(unit 8) is labelled or “tagged” using any one of a number of well-knowntechniques to introduce, for example, a radiochemical, fluorescent orbiotin label which will enable specific detection of saccharidescontaining the tag and, most importantly, which will provide a referencepoint or reading frame at the reducing end (RE) from which the sequencecan be read along the saccharide chain.

Use of a fluorescent compound to provide the label or tag will often bea preferred option, but when partial depolymerisation of the saccharidechain is subsequently to be carried out by low pH nitrous acid, as inthe present example, it is important to select a fluorescent compoundwhose fluorescence is not likely to be quenched by the nitrous acid.Thus, although it has been found that high coupling efficiencies can beachieved using an aminocoumarin hydrazide reagent (e.g.7-amino4-methylcoumarin-3-acetyl hydrazide, available from Pierce Ltd.U.K.) as a fluorescent label or tag, unfortunately it has been foundthat this is unsatisfactory for this present example because thefluorescence is quenched by nitrous acid. This labelling reagent,however, should be quite satisfactory for use when alternative selectivescission reagents are employed to carry out the partialdepolymerisation.

In the present case, therefore, the preferred fluorescent tag to use isanthranilic acid (2-aminobenzoic acid: ABA: excitation maxima, 290 nm,emission maxima, 390 nm) which is convenient and reasonably efficient.This reagent can be coupled specifically to the reducing end of sugarsby reductive animation as described previously by K. R. Anumula (1994)Analytical Biochemistry, 220, 275-283, but with some modifications asdescribed below.

For sulphated saccharides from GAGs at least, the following reactionconditions have been found to be satisfactory. The saccharides to becoupled (20-100 nmoles) are dried down in a microcentrifuge tube bycentrifugal evaporation, dissolved directly in 250 μL of formamidecontaining 200 mM ABA and 100 mM reductant (sodium cyanoborohydride),and heated at 50° C. for 16-24 hours.

After coupling free ABA, reductant and formamide can conveniently beremoved from tagged saccharides by methods such as dialysis, weak anionexchange chromatography or gel filtration chromatography. The latter isgenerally preferred since it usually allows quantitative recoveries ofloaded sample. The following procedure has been found to be convenient.The sample (250 μL of reaction mixture diluted to a total of 1 mL withdistilled water) is loaded onto two 5 mL HiTrap™ Desalting columns(products of Pharmacia Ltd). These are connected in series and elutedwith distilled water at a flow rate of 1 mL/min. Fractions of 0.5 mL arecollected. Saccharides consisting of 4 or more monosaccharide unitstypically elute in the void volume (approximately fractions 7-12). Thesefractions are pooled and concentrated by freeze drying or centrifugalevaporation. This approach allows rapid purification of taggedsaccharide material from free tagging reagent, gives quantitativerecoveries and the product is free of salts which might interfere withsubsequent enzymic conditions.

It has been observed that die fluorescence spectra of saccharide-ABAconjugates is modified as compared to that of the free ABA. Typicallythe conjugates display an excitation maxima in the range 300-320 nm ,which is ideal for visualistion with commonly available 312 nm UVsources (e.g. lamp or transilluminator). Emission maxima are typicallyin the range 400-420 nm (bright violet fluorescence).

Following coupling with fluorescent tags the saccharides can be furtherpurified if required prior to sequencing using techniques such as anionexchange HPLC or gradient PAGE. The latter has been found to beparticularly useful for purification purposes since it allows excellentresolution of tagged saccharides which can be readily recovered byelectrotransfer to positively charged nylon membrane as described forexample by Turnbull sand Gallagher in Biochemical Journal (1988) 251.597-608 and, with additional modifications, in Biochemical Journal(1991) 265. 715-724. Again, the content of these publications isincorporated herein by reference. In this technique the appropriatebands in the electrophoresis gel are cut out, their position beingestablished using a UV lamp (254 or 312 nm wavelength). The saccharidescan be dissociated from the membrane by incubation in 5M sodium chloridesolution in a microcentrifuge tube on a rotating mixer at 37° C. for 5hours, and can then be desalted by chromatography of the solution onHiTrap™ Desalting columns as described above. This approach isparticularly useful for preparing saccharides for sequencing fromsamples which are not purified to homogeneity prior to the fluorescenttagging step. Indeed, since gradient PAGE resolves many saccharides moreeffectively than other methods (for example anion exchange HPLC), it isthe method of choice for preparing homogeneous saccharide species forsequencing.

Upon treating the dp8 fragment illustrated in FIG. 1A with pHNO₂ ashereinbefore described a mixture of end-labelled or tagged fragments(herein referred to as pHNO₂ fragments) each with newly exposed NRE willbe produced, as illustrated in FIG. 1B of the accompanying drawings.

In the next stage, samples, preferably aliquots, of the mixturecontaining the pHNO₂ fragments are treated separately with differentspecific enzymes (singly or in combination) to remove accessiblesulphate groups and sugar residues, and the resulting saccharides arethen separated by gradient polyacryiamide gel electrophoresis (gradientPAGE) carried out in respect of each portion. This results in a bandingpattern that may be visualized as indicated in the diagram of FIG. 2 inthe accompanying drawings.

For simplicity this particular example describes only the use of enzymesto remove the terminal sulphated and non-sulphated hexuronates, but inpractice a small number of additional treatments may be needed toachieve a complete sequence identification. Samples of the pHNO₂fragments are treated separately with iduronate-2-sulphatase,glucuronidase and iduronidase and with the combination ofiduronate-2-sulphatase plus iduronidase. These enzyme treated samplesare each analysed separately in different tracks of the gradient PAGEseparation. Thus in the chart or diagram of FIG. 2 tracks c-f representthe enzyme treated samples, track b represents the separation of thecomplete set of pHNO₂ fragments I, II and III (FIG. 1B) without enzymetreatment, and the original fragment alone (Fragment I) is shown intrack a. This read-out then allows the majority of the sequence ofFragment I to be read. The manner of detection of the fragments will ofcourse depend on the nature of the tag, but will most commonly be byfluorescence and fluorographic methods, or by a calorimetric methodusing for example a biotin/avidin detection technique as known in theart.

To summarise, in FIG. 2 the identity of the samples in the differenttracks is as follows:

a) Fragment I

b) Partial HNO₂ hydrolysate of fragment I (pHNO)

c) pHNO₂+iduronate-2-sulphatase (I2Sase)—band shift marker*

d) pHNO₂+glucuronidase (Gase)—band shift marker $

e) pHNO₂+iduronidase (Idase)—band shift marker ∞

f) pH₂+iduronate-2-sulphatase+iduronidase—band shift markers σ and τ

Running conditions for such gel electrophoresis may be as describedpreviously in the literature, e.g. Turnbull and Gallagher (1988) BiochemJ. 251, 597-608. The migration banding pattern depicted in FIG. 2reflects tile different mobilities of saccharides with 2, 4, 6 and 8sugar units (dp2-8).

Further Detailed Description Applicable to Example 1 of the Treatment ofFluorescent Tagged Saccharides With Exoenzymes and of Separation of theTreated Saccharides by Page

As described above, in treating tagged saccharides generated by partialHNO₂ hydrolysis (pHNO₂) with exoenzymes, the mixed set of saccharides isdivided into an appropriate number of aliquots (one for each set ofexoenzyme digestion conditions and one left untreated). Thus, a pHNO₂treated sample of final volume 125 μL may be divided into 5 aliquots of25 μL, allowing sufficient for 4 different exoenzyme treatments. Eachaliquot would contain approximately 200-400 pmoles of saccharide.Treatment with exoenzymes requires addition of 10 μL of exoenzyme buffer(200 mM sodium acetate buffer, pH 4.0), 2 μL of 2 mg/mL bovine serumalbumin, 2 μL of appropriate enzyme (at concentrations of 3-6 U/ml where1U=1 μmole substrate hydrolysed per minute) and distilled H ₂O to bringthe final volume to 40 μL. The samples are then incubated at 37° C. for30-120 minutes which is usually sufficient to obtain complete digestion.The latter is important to the sequencing process since incompletedigestion would create a more complex banding pattern and would give afalse indication of sequence heterogeneity. Where combinations ofexoenzymes are required, these can be incubated sequentially orsimultaneously with the sample. Where an enzyme with a different pHoptima is used, an alternative buffer can be used both to terminate thepHNO₂ reaction and during setting up of the actual enzyme digestion. Ifnecessary, the activity of one enzyme can be destroyed prior to asecondary digestion with a different enzyme by heating the sample at100° C. for 1-5 minutes. Sample volumes can conveniently be reducedprior to electrophoresis by centrifugal evaporation.

The method of separating the treated saccharides for sequencing purposesby polyacryiamide gel electrophoresis (PAGE) is very effective and, asalready mentioned in connection with purification gradient PAGE isparticularly useful since it allows good resolution of a broad sizerange of saccharides on a single gel. The basic method, designatedoligosaccharide mapping has been described in detail as previouslyindicates by Turnbull and Gallagher (again see Biochemical Journal(1988) 251, 597-608 and Biochemical Journal (1991) 265, 715-724).Briefly, gradient PAGE gels, typically 16-32 cm in length and 0.5-3 mmin thickness, comprising a long resolving gel (with gradients of totalacrylamide concentrations in the range T20-50% and cross-linker rangingfrom C0.5% to C5%) and a short stacking gel (typically T5% acrylamide)are prepared using the buffer system described above. Samples (typically10-20 μL in 10% glycerol) are loaded into wells in the stacking gel andrun into the gel at 150 volts for 30 minutes, followed byelectrophoresis at 200-1000 volts until the run is complete (i.emigration of marker dyes to predetermined positions). Visualisation ofresolved fluorescent ABA-saccharide conjugates (picomole amounts) isreadily achieved using a UV transilluminator (312 nm wavelength). Theyappear as sharp bright violet fluorescent bands easily visible to thenaked eye. Improved sensitivity can be achieved using a charge coupleddevice (CCD) camera.

For sequencing purposes the gel should be loaded with a sample of intacttagged saccharide and of pHNO₂ generated saccharides not treated withexoenzymes, as well as the pHNO₂ samples actually treated withappropriate exoenzyme combinations. This allows the running position ofthe intact saccharide and pHNO₂ generated saccharides to be compareddirectly with the exoenzyme-treated saccharides (as shown in FIG. 2).

Interpretation of Migration Banding Pattern in FIG. 2

The migration banding pattern in FIG. 2 will next be described in moredetail. Track (a) illustrates the size of the original fragment andtrack (b) shows the banding pattern after the initial pHNO₂ treatment.The presence of two additional bands at dp6 and dp4 identifies GlcNSO₃residues at positions (2) and (4) in Fragment I (FIG. 1). Nodisaccharide band is seen so it can be deduced that the amino sugar atposition (6) is GlcNAc. Since GlcNAc must be α1,4 linked to GlcA, it canalso be deduced that this latter residue is present at position (7).After treatment with iduronate-2-sulphatase (track c) it is seen thatonly the original fragment shifts position (band *) thereby indicating a2-O-sulphate group on unit (1). Since β-glucuronidase (track d) onlyshifts the position of the band ($) representing Fragment II (dp6), thisidentifies GlcA as being the residue at position (3). Iduronidase (tracke) causes a shift only in the band (∞) representing Fragment III , sounsulphated iduronate is at position (5). Finally, the combination ofiduronate-2-sulphatase and iduronidase (track f) causes a shift inmobility of both Fragment III (τ) and Fragment I (σ). In the latter casethe shift in mobility exceeds that with the sulphatase enzyme alone(track c; band *). This confirms that the sugar residue at position (1)is iduronate-2-sulphate which becomes accessible to iduronidase afterenzymic removal of the 2-O-sulphate group. The banding pattern in FIG. 2thus enables the following features of the sequence of Fragment I to beread.

6-Sulphation at GlcNSO₃ or GlNAc

The presence of the 6-O-sulphate groups on the amino sugars at units (4)and (6) in Fragment I (FIG. 1) cannot be identified from the particularbanding pattern illustrated in FIG. 2. However, 6-O-sulphation of aminosugars can be easily detected by introducing an extra track (not shown)in which a further portion of the mixture of the pHNO₂ fragments istreated with a combination of the iduronate-2-sulphatase, iduronidaseand glucuronidase exoenzymes to ensure removal of the end chainhexuronates, together with glucosamine 6-O-sulphatase which will removethe 6-O-sulphate groups from the amino sugars now exposed at the ends ofthe fragments. The loss of 6-O-sulphate would be picked up by a mobilityshift on gradient PAGE. Consider, for example, the 6-O-sulphate at unit4 (FIG. 1). This would be present in Fragment II (dp6) after pHNO₂(track b in FIG. 2). Enzymic removal of the terminal GlcA in thisFragment II produces a dp5 fragment (track d; symbol $). This structurehas an exposed GlcNSO₃(6S) (unit 4) at the non-reducing end (NRE) andthe 6-O-sulphatase enzyme would then remove the 6-O-sulphate causing afurther increase in mobility.

Additional Approaches for Sequencing Contiguous N-acetylated Sequences

When sequencing HS/heparin saccharides there will sometimes be caseswhere one or more N-acetylated disaccharides intervene within anotherwise N-sulphated disaccharide sequence. This means that the pHNO₂treatment cannot create a new reducing end for exoenzyme attack and thiswould limit the sequence information which can be obtained at somepositions. For example, Fragment I in the present example contains aGlcNAc residue at position 6, and therefore pHNO₂ does not create afragment corresponding to positions 7 and 8. This means that theseresidues will not be directly sequenced. However, this problem can beovercome using the exoenzyme N-acetylglucosaminidase. Fragment IIIproduced by pHNO₂ can be treated with iduronidase (to remove theiduronic acid residue at position 5), glucosamine-6-sulphatase (toremove tie 6-O-sulphate on the GlcNAc at position 6) and thenN-acetylglucosaminidase (to remove the GlcNAc residue at position 6).This would result in a fragment corresponding to positions 7 and 8 (i.e.GlcA-GlcNSO₃*) and would allow the sequencing of the residues at thesepositions as already described as if the terminal uronate residue atposition 7 had been created by the pHNO₂ treatment. If more than oneGlcNAc residue intervenes, this process can be reiterated any number oftimes. Based on what is currently known about the structure of heparansulphate, in such a case the sequence is likely to consist of repeatingGlcA-GlcNAc residues predominantly without O-sulphate substitutents. Itwould thus be necessary to deactivate the glucuronidase orN-acetylglucosaminidase after each individual digestion to allow eachshift to be individually identified (i.e. to prevent a contiguoussequence of such residues being degraded in a single step as would occurwith a combination of both enzymes).

Sequence Microheterogenicity

It is also possible that two closely-related structures will run as asingle band on gradient PAGE. The sequencing strategy described wouldhowever detect this type of variability in sequence. Imagine for examplethat in position (3) some chains contained IdoA rather than GlcA—therewould then be both hexa and penta bands after the glucuronidase (trackd) and iduronidase (track e) treatments in proportion to the frequencyof occurrence at position (3). A track in which both enzymes Gase andIdase are used would effect a complete shift in the bands from hexa topenta and this would be clearly apparent.

There could also be variation in the sequence of N-sulphated (GlcNSO₃)and N-acetylated (GlcNAc) glucosamine residues. This could be detected,however, by use of N-acetylglucosaminidase which acts only onnon-reducing end (NRE) unsubstituted GlcNAc units. If for example, theGlcNSO₃ unit at position (2) was occasionally GlcNAc this could bedetected by running an extra track of the pHNO₂ saccharide mixtureincubated with an iduronidase, I2Sase, and Gase cocktail, heatinactivating the enzymes then incubating with N-acetylglucosaminidase.If unit (2) is always GlcNSO₃ there will be no reduction in molecularsize beyond dp7. The presence, however, of GlcNAc in a proportion of thesaccharides would then be revealed by an extra band at dp6 (hexa).

Further Options for Sequencing

HS and heparin may also contain O-sulphate groups at C-3 of GlcNSO₃— andand at C-2 of GlcA units. However exosulphatase enzymes are known thatcan specifically remove these substituents (see for example Irwin G.Leder (1980), “A novel 3-O sulfatase from human urine acting onmethyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulphate”, Biochemicaland Biophysical Research Communications, 94, 1183-1189; Ulf Lindahl etal, (1980), “Evidence for a 3-O-sulfated D-glucosamine residue in theantithrombin-binding sequence of heparin”, Biochemistry, 77, 6551-6555;Craig Freeman and John J Hopwood (1989) “Human liver glucuronate2-sulphatase”. Biochem. J., 259, 209-216 and Craig Freeman and John JHopwood (1992) “Human α-L-iduronidase”, Biochem. J., 282, 899-908).These enzymes may therefore also be incorporated into the sequencingstrategy. Likewise, (exo)N-sulphamidase (see for example Craig Freemanand John J Hopwood (1986)“Human Liver Sulphamate sulphohydrolase”,Biochem. J., 234, 83-92) could be used to confirm the presence ofN-sulphate groups.

As previously indicated a number of alternatives to pHNO₂ are alsoavailable for the initial partial depolymerisation and specific cleavageof internal linkages. Examples of known alternative reagents ortreatments include hydrazinolysis followed by treatment at pH 4.0 withHNO₂ (cleaves GlcNAc→HexA linkages), and also use of the endoenzymesheparitinase (cleaves GlcN.R→GlcA linkages) and heparinase (cleavesGlcNSO₃(±6S)→IdoA(2S) linkages). These latter lyase enzymes can alsoprovide valuable sequence information on the nature of the hexuronateresidues. Further sequencing of heparinase/heparitinase fragments mayrequire removal of the NRE unsaturated HexA(±2S) residue generated bythe endolytic mode of these enzymes which involves an eliminativecleavage mechanism, but this can be easily achieved by treatment withspecific bacterial enzymes (glycuronate sulphatase and glycuronidase) ormercuric salts (see for example U. Ludwigs et al (1987) “Reaction ofunsaturate uronic acid residues with mercuric salts” Biochem. J., 245.795-804).

In principle the method of the present invention is applicable tosaccharide fragments of any size and in practice its effective rangewill be limited only by the resolving power of separation techniquescurrently available.

Moreover, as already mentioned, the principle of this sequencing methodis also applicable to sequence analysis of saccharides excised fromother glycosaminoglycans (GAG's), glycoproteins or other saccharidechain containing material. Since an N-acetylated amino sugar is presentas a component of all disaccharide units in GAG's, saccharides ofinterest can be cleaved at the GlcNAc/GalNAc hexosaminidic linkage bypartial hydrazinolysis/pH 4.0 HNO₂ to yield fragments that can then besequenced by use of appropriate exoglycosidases and exosulphatasesfollowing the procedures herein described. Alternatively partialscission can be achieved by GAG-specific enzymes (e.g. chondroitinase ACand ABC for chondroitin and dermatan sulphate and keratanase for keratansulphate). End-chain tagging and separation techniques would be similarto those described for HS/heparin.

EXAMPLE 2

By way of a further explanatory example there is shown in FIG. 3 apolyacryiamide gel electrophoresis banding pattern derived from somepreliminary studies, depicting some aspects of the exosequencing methodof the present invention applied to a ³⁵S metabolically-labelledhexasaccharide fragment having the following simple repeating structure:

A test sample of this fragment was first treated with HNO₂ underconditions designed to produce only hydrolysis and partialdepolymerisation of susceptible linkages. The resulting mixture of pHNO₂fragments (dp 6, 4 and 2) was then desalted by gel filtration andresolved on a 32.5-40% gradient PAGE gel, either intact (i.e. withoutfurther treatment) or after treatment with different combinations ofexoenzymes. Combination treatments were carried out sequentially in theorder shown.

The tracks indicated in FIG. 3 were as follows (BB and PR indicate therunning positions of bromophenol blue and phenol red marker dyes)

(1) Untreated pHNO₂ fragments

(2) Gase only

(3) I2Sase only

(4) I2Sase+Idase

(5) I2Sase+Idase+G6Sase

The pHNO₂ treatment (track 1) resulted in the expected major bands atthe dp6 and dp4 positions (arrowed) and it is the shifts in these bandsthat need to be observed for sequencing purposes. The dp6 arrowed bandcorresponds to the intact original fragment. The dp4 arrowed bandrepresents the structure

IdoA(2S)→GlcNSO³(6S)-→ldoA(2S)-→AMann^(R)(6S).

(n.b. in this particular example the dp2 products migrated off the geland are not therefore seen).

With regard to the exoenzyme treatments, the results show a clearstepwise removal of NRE residues. Gase has no effect (track 2). I2Saseacts to remove a 2-O-sulphate group from both the dp6 and dp4 bandsresulting in shifts (track 3, bands a and a′ respectively). Idase thenacts to remove an iduronic acid residue, resulting in penta- andtri-saccharide products (track 4, bands b and b′ respectively). G6Sasecan then act to remove a 6-O-sulphate group giving a further shift(track 5, band c). (NB: the removal of the 6-O-sulphate from thetrisaccharide b′ resulted in its loss from the pattern due either tomigration off the gel or possibly lack of retention on a nylon membraneto which the separated fragments were transferred from the gel forfluorographic detection of the radiolabelled material.

Although in this test example the saccharide material was notselectively labelled and end referenced, the foregoing practical resultsconfirm that pHNO₂ can be used for partial depolymerisation and thatexoglycosidases and exosulphatases act on NRE sugar residues to producethe predicted shifts in mobilities of oligosaccharide fragments.

Thus, these results, although only performed on a small test sample,clearly demonstrate not only that the exosequencing strategy can be usedto determine rapidly and unequivocally the sequence of monosaccharideresidues and sulphate groups at the NRE terminus of HS/Heparinfragments, but in addition they show that this general strategy can beapplied to newly created NRE terminii generated by partial internalcleavage of a fragment at GlcNSO₃ residues with a selective scissionreagent such as HNO₂.

Alternative Sequencing Strategy Involving Selective Coupling ofSaccharides to a Solid Phase Support

An alternative to tagging the chains of the saccharide material with adetectable labelling compound specifically at the reducing end forsequencing purposes is to couple or attach them to a solid-phase supportselectively via their reducing ends. The partial internal cleavage (e.g.pHNO₂) of glycosidic linkages can then be carried out whilst thesaccharide chains are thus immobilized and fragments which are no longercontiguous with the reducing end can be easily removed by thoroughwashing. Provided a method is available then to release the saccharidesattached at their reducing ends from the solid phase support, a mixedset of saccharide chain fragments equivalent to those created by pHNO₂treatment of fluorescent tagged saccharides is obtained. In effect, thisagain provides a reducing end referencing feature. Such an approach hasbeen described previously to obtain “end-referenced” polysaccharidechains by Radoff and Danishefsky (1984), J. Biol. Chem., 259, 166-172who attached a coupling agent (tyramine) to the reducing end terminus ofheparin saccharide chains for coupling to an insoluble activatedSepharose™ matrix.

In this method the saccharide chains of the saccharide material willusually be selectively modified by first attaching as a tag to theirreducing ends a coupling agent for the coupling to the solid phasesupport. However, in the case of sequencing the saccharide component ofmaterial such as proteoglycans for example where the reducing ends ofthe saccharide chains are already joined or conjugated to polypeptidechains, no initial modification may be needed as these existingpolypeptide chains may be used directly to couple to on appropriatesolid phase matrix support, e.g. CNBr-activated Sepharose 4B™, asdescribed for example by Lyon et al (1987) Biochem. J. 242, 493-498 andby Turnbull and Gallagher (1991) Biochem. J. 277, 297-303).

This approach involving immobilizing the saccharide material by couplingto a solid phase support can be particularly suited to carrying out thesequence analysis method of the present invention on small amounts ofsample (for example, as from cultured cells) radiolabelledbiosynthetically (for example with 3H-glucosamine).

In practice, the procedure may also be modified slightly in that thepartial depolymerisation step may be carried out before coupling andimmobilizing the saccharide chains or fragment thereof on the solidphase support. For example, the saccharides can first be taggedspecifically at the reducing ends with a coupling agent in the form of2-imino-biotin hydrazide, and then they can be subjected to pHNO₂treatment as already described. After the pHNO₂ treatment the fragmentscan then be captured on avidin-agarose by virtue of the reducing endbiotin residues. Following thorough washing, the saccharides remainingattached can be dissociated from the gel under mild conditions byeluting the gel with a pH 4 buffer. The recovered saccharides can thenbe sequenced directly, again as already described. After electrophoresisthey can be conveniently detected by electrotransfer to nylon membranematerial and fluorography, again as described in the literature (seeagain Turnbull and Gallagher in Biochemical Journal (1988) 251, 597-608and in Biochemical Journal (1991) 265. 715-724).

As will be seen, the invention provides a number of different aspectsand. in general it embraces all novel and inventive features and aspectsherein disclosed either explicitly or implicitly and either singly or incombination with one another. Moreover, the scope of the invention isnot to be construed as being limited by the. illustrative examples or bythe terms and expressions used herein merely in a descriptive orexplanatory sense, and many modifications may be made within the scopeof the invention defined in the appended claims.

What is claimed is:
 1. A method of analyzing and sequencing saccharidematerial composed of saccharide chains having more than threemonosaccharide units interconnected through glycosidic linkages that arenot all identical, wherein each saccharide has a referencing feature atits reducing end, said method comprising the sequential steps of: (a)treating said saccharide material in a partial depolymerisation stagewith a selective scission reagent that acts in accordance with apredetermined linkage specificity as an endoglycosidase to substantiallycleave internal glycosidic linkages spaced from the non-reducing end ofthe saccharide chains, thereby producing a mixed set of saccharidechains comprising intact chains and fragments of intact chains havingdifferent lengths representative of the full spectrum of all possiblelengths given the particular glycosidic linkage specificity of theselective scission reagent employed, (b) treating a sample or samples ofsaid mixed set of saccharide chains and chain fragments from the partialdepolymerization treatment of step (a) with a set of exoenzymes whichincludes exoglycosidases of known specificity that cleave onlyparticular glycosidic linkages at the non-reducing end of saccharidechains, said exoenzymes being applied, either singly or in combination,in accordance with a predetermined strategy, (c) continuing step (b) toan extent sufficient to obtain complete digestion and cleave susceptiblelinkages at the non-reducing end of all the saccharide chains, and then,(d) analyzing said sample or samples to detect the saccharide chainfragments generated by cleavage treatments, said fragments having areducing end derived from the reducing end of the corresponding chain inthe original saccharide material, and at least partially deducing themonosaccharide sequence in the saccharide material.
 2. A method asclaimed in claim 1, wherein said saccharide chains have attached totheir reducing ends a detectable label or tag providing a reducing endreference feature, wherein said label or tag is selected from the groupconsisting of a radiochemical labeling agent, a fluorescent labelingagent and a calorimetrically detectable labeling agent.
 3. A method asclaimed in claim 2 wherein the detectable label or tag of the reducingend referencing feature comprises a compound selected from the groupconsisting of anthranilic acid, an aminocoumarin hydrazide and atritiated borohydride.
 4. A method as claimed in claim 1 furthercomprising, before step (a), a step of modifying said saccharide chainsby attaching to their reducing ends a tag comprising a coupling agentfor immobilizing said chains or reducing end fragments thereof bycoupling to a solid phase support, while fragments of said chainsproduced by the partial depolymerization treatment which are notcontiguous with the reducing ends of original undegraded chains areseparated and removed.
 5. A method as claimed in claim 4 including thestep of coupling the saccharide chains to a said solid phase support toimmobilize said chains, followed by a step of separating and removingfragments of said chains produced by the partial depolymerizationtreatment which are not contiguous with the reducing ends of theoriginal undegraded chains, followed by the step of releasing residualimmobilized saccharide chain fragments from said solid phase supportprior to the exoenzyme treatment of step (b).
 6. A method as claimedclaim 1 wherein the selected exoenzymes further include specificexosulphatases for selective removal of sulphate groups frommonosaccharide residues at the non-reducing end of said saccharidechains or fragments of said chains.
 7. A method as claimed in claim 1wherein the selective scission reagent used in step (a) for thecontrolled partial depolymerization of the saccharide material is amember selected from the group consisting of nitrous acid andendoglycosidase enzymes.
 8. A method as claimed in claim 7 wherein saidselective scission reagent is nitrous acid introduced to said saccharidematerial under low pH conditions whereby said acid specifically cleaveshexosaminidic linkages that link an N-sulphated amino sugar to ahexuronate residue but not hexosaminidic linkages that link anN-acetylated amino sugar to a hexuronate residue.
 9. A method as claimedin claim 1 wherein the partial depolymerization treatment of step (a) iscarried out by treating separate samples of the saccharide material withsaid selective scisson reagent for different periods of time and thenpooling the products to provide the mixed set of chain fragments for usein step (b), thereby ensuring that the lengths of the saccharide chainfragments in said mixed set of chain fragments used in step (b) aredistributed throughout the range extending from full length undegradedchains to minimum length chain fragments lacking in further internalglycosidic linkages cleavable by said selective scission reagent.
 10. Amethod as claimed in claim 1 in which one sample of the mixed set ofsaccharide chain fragments produced by the partial depolymerization ofthe saccharide material in step (a) is subjected without exoenzymetreatment to a separation procedure to separate said chain fragmentsaccording to length, and other samples are also subjected, separately,to the same said separation procedure to separate the saccharide chainfragments therein according to length and composition after each ofthese other samples has been treated with a different exoenzyme orcombination of exoenzymes in step (b).
 11. A method as claimed in claim1 wherein the analysis of step (c) includes separating the saccharidechain fragments produced by the cleavage treatments according to theirlength and composition.
 12. A method as claimed in claim 11 wherein theseparated saccharide chain fragments that include the reducing endresidues of the original saccharide chains of the saccharide materialare detected by detecting a label or tag carried by the separatedsaccharide chain fragments.
 13. A method as claimed in claim 11 whereinsaid separation of the saccharide chain fragments is effected by anelectrophoretic separation technique whereby the saccharide fragmentsare separated according to differences in length and composition whichresult in different mobilities in the electrophoretic medium.
 14. Amethod as claimed in claim 13 which comprises treating separate samplesof the mixed set of saccharide chain fragments from step (a) withdifferent said exoenzymes, either singly or in combination, carrying outstep (b) and subjecting said samples simultaneously in step (c) toelectrophoretic separation in different tracks of an electrophoresisgel, thereby providing a migration banding pattern representative of thedifferent gel mobilities of the separated fragments having differencesin length and/or composition.
 15. A method as claimed in claim 14wherein the electrophoretic separation is carried out by gradientpolyacryiamide gel electrophoresis (gradient PAGE).
 16. A method asclaimed in claim 14 which includes transferring the separated saccharidechain fragments by an electrotransfer technique to a synthetic polyamidemembrane after termination of the electrophoresis.
 17. A method asclaimed in claim 1 wherein labeled or “tagged” fragments that includethe reducing and residues of the original saccharide chains of thesaccharide material provide in the analysis of step (c) avisually-detectable pattern which, in conjunction with the knownspecificity of the exoenzymes used in step (b), gives sequenceinformation directly by deduction.
 18. A method as claimed in claim 1 inwhich the saccharide material is composed of saccharide chainscontaining amino sugar residues and sulphated monosaccharide units, andthe exoenzymes used in step (b) are selected from the group consistingof a glucuronidase, an iduronidase, an N-acetylglucosaminidase, aniduronate-2-sulphatase, a glucuronate-2-sulphatase, aglucosamine-6-sulphatase, a glucoasmine-3-sulphatase and a sulphamatesulphohydrolase.